The extracellular loops of and ? ENaC can be roteolytically cleaved at multiple sites by furin-type convertases, serine proteases and neutrophil elastase, leading to activation of the channel and increased Na+ absorption. In cystic fibrosis (CF) airways, ENaC is abnormally hyperactive due in part to excessive proteolytic cleavage, which contributes to a depletion of airway surface liquid (ASL) volume. This in turn may lead to mucus stasis and increased incidence of airway infections that frequently lead to the death of the patient. We have recently identified SPLUNC1 as a potent inhibitor of ENaC that binds to the extracellular side of ENaC and diminishes plasma membrane ENaC levels. We have also shown that when SPLUNC1 is knocked down by shRNA, normal human bronchial epithelial cultures can no longer regulate ENaC activity. We hypothesize that SPLUNC1 limits Na+ absorption by reducing ENaC surface densities, preventing exposure to extracellular proteases. We have identified the active site of SPLUNC1, and have synthesized an 18 amino acid peptide based on this site, called S18, which specifically binds to ENaC. Although S18 robustly inhibits ENaC in NL and CF airway epithelia, SPLUNC1 fails to regulate ENaC in CF airway epithelia. CF ASL is acidic due to the lack of CFTR-associated HCO3- secretion. Molecular modeling indicates that SPLUNC1 may undergo a pH- induced conformational change that buries its active site, preventing binding to ENaC in the acidic CF ASL. Thus, we propose to (i) solve the crystal structure of SPLUNC1 to further refine this model, (ii) determine the mechanism whereby extracellular SPLUNC1 can reduce ENaC surface density and (iii) determine why SPLUNC1 fails to function in CF airways. S18-like peptides may be therapeutically beneficial in the treatment of CF lung disease. Furthermore, our data suggest that inhaled hypertonic HCO3- may offer significant advantages over hypertonic saline for restoring CF mucus clearance.